Target-Dependent Regulation of Neurotransmitter Specification and Embryonic Neuronal Calcium Spike Activity

Abstract

Neurotransmitter specification has been shown to depend on genetic programs and electrical activity; however, target-dependent regulation also plays important roles in neuronal development. We have investigated the impact of muscle targets on transmitter specification in Xenopus spinal neurons using a neuron-muscle coculture system. We find that neuron-muscle contact reduces the number of neurons expressing the noncholinergic transmitters GABA, glycine, and glutamate, while having no effect on the incidence of ChAT expression. We show that muscle activity is necessary for target-dependent reduction of noncholinergic transmitter expression. In addition, we demonstrate that coculture with muscle cells suppresses early spontaneous calcium spike activity in neurons and the presence of muscle cells abolishes activity-dependent transmitter specification. The results indicate that target-dependent regulation can be crucial in establishing neurotransmitter phenotypes and altering early neuronal excitability.

Calcium Activity Dynamics Correlate with Neuronal Phenotype at a Single Cell Level and in a Threshold-Dependent Manner

“…Given the non-canonical and irregular nature of calcium activity in the early, embryonic nervous system as well as the lack of a consistent definition of what constitutes a spike in the current literature, we theorized that the application of different analysis methods and threshold parameters could dramatically affect the conclusions drawn from a dataset. Different groups have defined a spike as: (i) an instance of fluorescence intensity greater than 150% of the baseline (dF/F0) [34]; (ii) a transient amplitude with a rise time of 5 s reaching more than 20% of the baseline value (dF/F0) as determined by the values over the previous 10 min [39]; (iii) a transient amplitude with a rise time of 5 s that exceeds twice the baseline variation during the previous 10 min [46]; and (iv) any transient lasting less than 3 min and with an amplitude of more than three standard deviations above the average value (>3 SD) [35]. Furthermore, the method of baseline establishment differs between even between these reports, complicating any attempt to synthesize conclusions from the current body of published literature.…”

Calcium Signals Regulate Neurotransmitter Phenotype

“…Musclederived factors play important roles in many aspects of neuronal development, including neuronal survival, axon and dendrite outgrowth, neuronal differentiation, and synaptogenesis (Zweifel et al, 2005). When muscle cells are omitted from cultures, neurons exhibit a much higher spike frequency (Xiao et al, 2010;Figure 56.3(c)). When muscle cells are omitted from cultures, neurons exhibit a much higher spike frequency (Xiao et al, 2010;Figure 56.3(c)).…”

Dissection, Culture, and Analysis of <em>Xenopus laevis</em> Embryonic Retinal Tissue

“…For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,[19][20][21][22][24][25][26][27][28][29][30][31][32][33] . Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] .…”

“…Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] . Xenopus laevis, a classic model system for the study of early neural development 19,27,29,[31][32][40][41][42] , serves as a particularly suitable system for retinal primary cell culture 10,38,[43][44][45] .…”